Neutralization of interleukin-18 ameliorates ischemia/reperfusion-induced myocardial injury.
ABSTRACT: Ischemia/reperfusion (I/R) injury is characterized by the induction of oxidative stress and proinflammatory cytokine expression. Recently demonstrating that oxidative stress and TNF-alpha each stimulate interleukin (IL)-18 expression in cardiomyocytes, we hypothesized that I/R also induces IL-18 expression and thus exacerbates inflammation and tissue damage. Neutralization of IL-18 signaling should therefore diminish tissue injury following I/R. I/R studies were performed using a chronically instrumented closed chest mouse model. Male C57BL/6 mice underwent 30 min of ischemia by LAD coronary artery ligation followed by various periods of reperfusion. Sham-operated or ischemia-only mice served as controls. A subset of animals was treated with IL-18-neutralizing antibodies 1 h prior to LAD ligation. Ischemic LV tissue was used for analysis. Our results demonstrate that, compared with sham operation and ischemia alone, I/R significantly increased (i) oxidative stress (increased MDA/4-HNE levels), (ii) neutrophil infiltration (increased MPO activity), (iii) NF-kappaB DNA binding activity (p50, p65), and (iv) increased expression of IL-18Rbeta, but not IL-18Ralpha or IL-18BP transcripts. Administration of IL-18-neutralizing antibodies significantly reduced I/R injury measured by reduced infarct size (versus control IgG). In isolated adult mouse cardiomyocytes, simulated ischemia/reperfusion enhanced oxidative stress and biologically active IL-18 expression via IKK-dependent NF-kappaB activation. These results indicate that IL-18 plays a critical role in I/R injury and thus represents a promising therapeutic target.
Project description:BACKGROUND:Diabetic patients are susceptible to renal ischemia-reperfusion injury, which leads to perioperative complications. Activation of NOD-like receptor protein 3 (NLRP3) inflammasome participates in the development of diabetes, and contributes to renal ischemia-reperfusion injury. Dexmedetomidine (DEX), a highly selective ?2-adrenoreceptor agonist, shows renoprotective effects against ischemia-reperfusion injury. We aimed to elucidate the effects, underlying mechanisms, and optimal timing of DEX treatment in diabetic rats. METHODS:Male Sprague-Dawley rats (n = 12 per group) were randomly divided into normal-sham, diabetes-sham, diabetes-ischemia-reperfusion-control, diabetes-ischemia-reperfusion-DEX-pre-treatment, and diabetes-ischemia-reperfusion-DEX-post-treatment groups. Renal ischemia-reperfusion injury was induced in diabetic rats by occlusion of both renal arteries for 45 min, followed by reperfusion for 24 h. DEX (10 ?g/kg) was administered intraperitoneally 1 h before ischemia (pre-treatment) or upon reperfusion (post-treatment). After reperfusion, renal tissue was biochemically and histopathologically evaluated. RESULTS:DEX treatment attenuated ischemia reperfusion-induced increase in NLRP3, caspase-1, IL-1?, phospho-AKT, and phospho-ERK signaling. Moreover, oxidative stress injury, inflammatory reactions, apoptosis, and renal tubular damage were favorably modulated by DEX treatment. Furthermore, post-reperfusion treatment with DEX was significantly more effective than pre-treatment in modulating NLRP3 inflammasome, AKT and ERK signaling, and oxidative stress. CONCLUSIONS:This study shows that the protective effects of DEX in renal ischemia-reperfusion injury are preserved in diabetic conditions and may potentially provide a basis for the use of DEX in clinical treatment of renal ischemia-reperfusion injury.
Project description:Mice were exposed to cardiac ischemia/reperfusion injury and sacrificed after 1 or 4 days. Hearts were stained with Evans blu/TTC to isolate the at risk area. Hearts were divided into 1mm slices, the at risk area of any slice were pooled together before RNA extraction. Mice were divided into 3 different groups: Sham animals have been subjected to the surgical procedure without the induction of the ischemia and reperfusion injury and represent the control animals; LAD group is represented by animals subjected to the surgical procedure and the induction of the ischemia and reperfusion imjury; MLAD group is represented by animals subjected to the surgical procedure and the induction of the ischemia and reperfusion injury, followed by myriocin treatment (SLN/Myr) at the beginning of reperfusion. Overall design: qPCR gene expression profiling. Samples were obtained from 5 to 7 animals per group (Sham; LAD; MLAD). For any given target gene, the average expression value of all the sham animals has been compared to the expression value of each animal from LAD and MLAD group to calculate their relative expression levels. Statistical analyses were performed comparing: LAD vs Sham; MLAD vs Sham; LAD vs MLAD at both time points of reperfusion.
Project description:Interleukin-4 (IL-4) has a protective effect against cerebral ischemia/reperfusion injury. Animal experiments have shown that IL-4 improves the short- and long-term prognosis of neurological function. The Akt (also called protein kinase B, PKB)/glycogen synthase kinase-3? (Akt/GSK-3?) signaling pathway is involved in oxidative stress, the inflammatory response, apoptosis, and autophagy. However, it is not yet clear whether the Akt/GSK-3? pathway participates in the neuroprotective effect of IL-4 against cerebral ischemia/reperfusion injury. In the present study, we established a cerebral ischemia/reperfusion mouse model by middle cerebral artery occlusion for 60 minutes followed by a 24-hour reperfusion. An IL-4/anti-IL-4 complex (10 ?g) was intraperitoneally administered 30 minutes before surgery. We found that administration of IL-4 significantly alleviated the neurological deficits, oxidative stress, cell apoptosis, and autophagy and reduced infarct volume of the mice with cerebral ischemia/reperfusion injury 24 hours after reperfusion. Simultaneously, IL-4 activated Akt/GSK-3? signaling pathway. However, an Akt inhibitor LY294002, which was injected at 15 nmol/kg via the tail vein, attenuated the protective effects of IL-4. These findings indicate that IL-4 has a protective effect on cerebral ischemia/reperfusion injury by mitigating oxidative stress, reducing apoptosis, and inhibiting excessive autophagy, and that this mechanism may be related to activation of the Akt/GSK-3? pathway. This animal study was approved by the Animal Ethics Committee of Renmin Hospital of Wuhan University, China (approval No. WDRY2017-K037) on March 9, 2017.
Project description:Objective Myocardial ischemia and reperfusion (I/R) injury is associated with oxidative stress and inflammation, leading to scar development and malfunction. The marine omega-3 fatty acids (?-3 FA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are mediating cardioprotection and improving clinical outcomes in patients with heart disease. Therefore, we tested the hypothesis that docosahexaenoic acid (DHA) supplementation prior to LAD occlusion-induced myocardial injury (MI) confers cardioprotection in mice. Methods C57BL/6N mice were placed on DHA or control diets (CD) beginning 7?d prior to 60?min LAD occlusion-induced MI or sham surgery. The expression of inflammatory mediators was measured via RT-qPCR. Besides FACS analysis for macrophage quantification and subtype evaluation, macrophage accumulation as well as collagen deposition was quantified in histological sections. Cardiac function was assessed using a pressure-volume catheter for up to 14?d. Results DHA supplementation significantly attenuated the induction of peroxisome proliferator-activated receptor-? (PPAR-?) (2.3 ± 0.4 CD vs. 1.4 ± 0.3 DHA) after LAD occlusion. Furthermore, TNF-? (4.0 ± 0.6 CD vs. 1.5 ± 0.2 DHA), IL-1? (60.7 ± 7.0 CD vs. 11.6 ± 1.9 DHA), and IL-10 (223.8 ± 62.1 CD vs. 135.5 ± 38.5 DHA) mRNA expression increase was diminished in DHA-supplemented mice after 72?h reperfusion. These changes were accompanied by a less prominent switch in ?/? myosin heavy chain isoforms. Chemokine mRNA expression was stronger initiated (CCL2 6?h: 32.8 ± 11.5 CD vs. 78.8 ± 13.6 DHA) but terminated earlier (CCL2 72?h: 39.5 ± 7.8 CD vs. 8.2 ± 1.9 DHA; CCL3 72?h: 794.3 ± 270.9 CD vs. 258.2 ± 57.8 DHA) in DHA supplementation compared to CD mice after LAD occlusion. Correspondingly, DHA supplementation was associated with a stronger increase of predominantly alternatively activated Ly6C-positive macrophage phenotype, being associated with less collagen deposition and better LV function (EF 14?d: 17.6 ± 2.6 CD vs. 31.4 ± 1.5 DHA). Conclusion Our data indicate that DHA supplementation mediates cardioprotection from MI via modulation of the inflammatory response with timely and attenuated remodeling. DHA seems to attenuate MI-induced cardiomyocyte injury partly by transient PPAR-? downregulation, diminishing the need for antioxidant mechanisms including mitochondrial function, or ?- to ?-MHC isoform switch.
Project description:Iron-mediated oxidative stress is implicated in the pathogenesis of renal ischemia-reperfusion injury. Hepcidin is an endogenous acute phase hepatic hormone that prevents iron export from cells by inducing degradation of the only known iron export protein, ferroportin. In this study, we used a mouse model to investigate the effect of renal ischemia-reperfusion injury on systemic iron homeostasis and determine if dynamic modulation of iron homeostasis with hepcidin has therapeutic benefit in the treatment of AKI. Renal ischemia-reperfusion injury induced hepatosplenic iron export through increased ferroportin expression, which resulted in hepatosplenic iron depletion and an increase in serum and kidney nonheme iron levels. Exogenous hepcidin treatment prevented renal ischemia-reperfusion-induced changes in iron homeostasis. Hepcidin also decreased kidney ferroportin expression and increased the expression of cytoprotective H-ferritin. Hepcidin-induced restoration of iron homeostasis was accompanied by a significant reduction in ischemia-reperfusion-induced tubular injury, apoptosis, renal oxidative stress, and inflammatory cell infiltration. Hepcidin -: deficient mice demonstrated increased susceptibility to ischemia-reperfusion injury compared with wild-type mice. Reconstituting hepcidin-deficient mice with exogenous hepcidin induced hepatic iron sequestration, attenuated the reduction in renal H-ferritin and reduced renal oxidative stress, apoptosis, inflammation, and tubular injury. Hepcidin-mediated protection was associated with reduced serum IL-6 levels. In summary, renal ischemia-reperfusion injury results in profound alterations in systemic iron homeostasis. Hepcidin treatment restores iron homeostasis and reduces inflammation to mediate protection in renal ischemia-reperfusion injury, suggesting that hepcidin-ferroportin pathway holds promise as a novel therapeutic target in the treatment of AKI.
Project description:Liver ischemia-reperfusion injury (IRI) is a common clinical event with high morbidity in patients undergoing complex liver surgery or having abdominal trauma. Inflammatory and oxidative stress responses are the main contributing factors in liver IRI. The iridoid glucoside aucubin (AU) has good anti-inflammatory and antioxidative effects; however, there are no relevant reports on the protective effect of glucosides on hepatic IRI. The purpose of this study was to determine whether AU pretreatment could prevent liver IRI and to explore the mechanism. Sprague-Dawley rats were randomly divided into five groups. The sham operation and IRI control groups were given intraperitoneal injections of normal saline, while the AU low-dose (AU-L) group, AU medium-dose (AU-M) group, and AU high-dose (AU-H) group were given intraperitoneal injections of AU at doses of 1, 5, and 10 mg/kg/day, respectively. After 10 d, liver IRI (70% liver ischemia for 1 h, reperfusion for 6 h) was surgically established in all groups except the sham group. Our results confirmed that liver injury was significantly aggravated after hepatic ischemia-reperfusion. AU alleviated the increase of transaminase and pathological changes induced by ischemia-reperfusion and improved liver damage. AU could also ameliorate the inflammatory and oxidative stress responses induced by ischemia-reperfusion and reduced expression of high mobility group protein (HMG)B1, receptor for advanced glycation end-products (RAGE), tumor necrosis factor (TNF)-?, interleukin (IL)-1?, and reactive oxygen species (ROS). Moreover, AU reduced ischemia-reperfusion-induced mitochondrial dysfunction and cells apoptosis, increased peroxisome proliferator-activated receptor ? coactivator (PGC)-1? and uncoupling (UCP)2 protein expression, and reduced caspase-3, cleaved caspase-3, and Cytochrome P450 proteins (CYP) expression. To determine expression levels of the Toll-like receptor (TLR)-4/nuclear factor-?B (NF-?B) pathway-related proteins in vitro and in vivo, we also measured TLR-4, myeloid differentiation factor88 (MyD88), NF-?B P65, p-P65, I-kappa-B-alpha (I?B-?), and p-I?B-? levels. The results showed that AU effectively inhibited activation of the TLR-4/NF-?B signaling pathway. In conclusion, we showed for the first time a hepatoprotective effect for AU in liver IRI, which acted by inhibiting the HMGB1/TLR-4/NF-?B signaling pathway, oxidative stress, and apoptosis. Pretreatment with AU may be a promising strategy for preventing liver IRI.
Project description:<h4>Background</h4>Our previous research showed the antioxidant activity of anthocyanins extracted from <i>Aronia melanocarpa</i> of black chokeberry in <i>vitro.</i> Ischemia acute kidney injury is a significant risk in developing progressive and deterioration of renal function leading to clinic chronic kidney disease. There were many attempts to protect the kidney against this progression of renal damage. Current study was designed to examine the effect of pretreatment with three anthocyanins named cyanidin-3-arabinoside, cyanidin-3-glucodise, and cyaniding-3-galactoside against acute ischemia-reperfusion injury in mouse kidney.<h4>Methods</h4>Acute renal injury model was initiated by 30?min clamping bilateral renal pedicle and followed by 24-hour reperfusion in C57Bl/6J mice. Four groups of mice were orally pretreated in 50?mg/g/12?h for two weeks with cyanidin-3-arabinoside, cyanidin-3-glucodise, and cyaniding-3-galactoside and anthocyanins (three-cyanidin mixture), respectively, sham-control group and the renal injury-untreated groups only with saline.<h4>Results</h4>The model resulted in renal dysfunction with high serum creatinine, blood urea nitrogen, and changes in proinflammatory cytokines (TNF-?, IL-1<i>?</i>, IL-6, and MCP-1), renal oxidative stress (SOD, GSH, and CAT), lipid peroxidation (TBARS and MDA), and apoptosis (caspase-9). Pretreatment of two weeks resulted in different extent amelioration of renal dysfunction and tubular damage and suppression of proinflammatory cytokines, oxidative stress, lipid peroxidation, and apoptosis, thus suggesting that cyanidins are potentially effective in acute renal ischemia by the decrease of inflammation, oxidative stress, and lipid peroxidation, as well as apoptosis.<h4>Conclusion</h4>the current study provided the first attempt to investigate the role of anthocyanins purified from <i>Aronia melanocarpa</i> berry in amelioration of acute renal failure via antioxidant and cytoprotective effects.
Project description:INTRODUCTION:Essential amino acid ?-keto acid analogs are used in the treatment of chronic kidney disease to delay the symptoms of uremia. However, it is unknown whether essential amino acid ?-keto acid analogs affect the oxidative stress and the inflammation in acute renal injury such as those produced by ischemia-reperfusion. OBJECTIVE:To evaluate the effect of essential amino acid ?-keto acid analogs on renal ischemia-reperfusion injury in Wistar rats. MATERIALS AND METHODS:Rats were divided into 11 groups (n=6/group): Two groups received physiological saline with or without ischemia-reperfusion injury (45 min/24 h), six groups received essential amino acid ?-keto acid analogs (400, 800, or 1,200 mg/kg/24 h/7d) with or without ischemia-reperfusion injury (essential amino acid ?-keto acid analogs + ischemia-reperfusion), and two groups received allopurinol (50 mg/kg/24 h/7d) with or without ischemia-reperfusion injury. Biochemical markers included creatinine and blood urea nitrogen (BUN), proinflammatory cytokines (IL-1?, IL-6, and TNF-?), renal damage markers (cystatin C, KIM-1, and NGAL), and markers of oxidative stress such as malondialdehyde (MDA) and total antioxidant activity. RESULTS:The essential amino acid ?-keto acid analog- and allopurinol-treated groups had lower levels of creatinine, BUN, renal damage markers, proinflammatory cytokines, and MDA than their corresponding ischemia-reperfusion groups. These changes were related to the essential amino acid ?-keto acid analogs dosage. Total antioxidant activity was lower in essential amino acid ?-keto acid analog- and allopurinol-treated groups than in the corresponding ischemia-reperfusion groups. CONCLUSIONS:This is a new report on the nephroprotective effects of essential amino acid ?-keto acid analogs against ischemia-reperfusion injury. Essential amino acid ?-keto acid analogs decreased the levels of biochemical markers, kidney injury markers, proinflammatory cytokines, and MDA while minimizing total antioxidant consumption.
Project description:Objectives:The aim of this study is to determine the therapeutic effects of pomegranate seed oil, which is a powerful antioxidant and anti-inflammatory agent, on ovarian-ischemia and reperfusion injury in rats. Materials and Methods:Fifty-six female albino Wistar rats were divided into 7 equal groups. Group 1; Sham Operation, Group 2; Ischemia, Group 3; Ischemia + Reperfusion, Group 4; Ischemia + Pomegranate 0,32 ml / kg (IP), Group 5; Ischemia + Pomegranate 0.64 ml / kg, Group 6; Ischemia + Pomegranate 0,32 ml / kg + reperfusion, Group 7; Ischemia + Pomegranate 0,64 ml / kg + reperfusion. Three hours after ischemia and 3 hours after reperfusion, the study was terminated. Results:While NADPH oxidase activity, MDA and TNF-? levels were significantly increased, SOD activity and GSH levels were reduced in ischemia and I/R groups. Low dose pomegranate seed oil application reduced significantly oxidative stress and NADPH oxidase activity in both ischemic and ischemic/reperfusion groups. At the same time, low-dose pomegranate seed oil extract reduced TNF-? levels and significantly increased antioxidant activity. Conclusion:PSO demonstrated an important therapeutic effect in the treatment of ovarian ischemia and reperfusion injury.
Project description:<h4>Background</h4>Despite a genomic revolution in biological sciences, clinical medicine has yet to integrate diagnostics based upon gene expression into practice. While commonly used plasma protein assays rely on organ-specific origins, nearly all nucleic acid in whole blood is derived from white blood cells limiting their utility to diagnose non-immune disorders. The aim of the study was to use cell-free plasma to define circulating messenger RNA sequences diagnostic of acute organ injury, including myocardial infarction (MI) and acute kidney injury (AKI).<h4>Methods</h4>In healthy human subjects (N?=?4) and patients with acute MI (N?=?4), we characterized the concentration and nature of circulating plasma RNA through spectrophotometry and chromatography. Through reverse transcriptase polymerase chain reaction (RT-PCR) of amplicons up to 939 base pairs, we determined whether this mRNA was intact but of insufficient quantity to sequence. In mice, we induced an acute anterior myocardial infarction through 1 h of ischemia followed by reperfusion of the left anterior descending (LAD) artery. We compared the cell-free plasma transcriptome using cDNA microarray in sham-operated mice compared to ischemia upon reperfusion and at 1 and 4 h. To determine organ specificity, we compared this profile to acute ischemia-reperfusion of the kidney.<h4>Results</h4>In humans, there is more plasma RNA in those with acute MI than in healthy controls. In mice, ischemia-reperfusion of the LAD artery resulted in a time-dependent regulation of 589 circulating mRNA transcripts with less than a 5% overlap in sequences from acute ischemia-reperfusion injury of the kidney.<h4>Conclusions</h4>The mRNA derived from cell-free plasma defines organ injury in a time and injury-specific pattern.